1. Field of the Invention
This invention pertains to production of recombinant proteins and more particularly to a means of heterologous gene transactivation.
2. Related Art
The ability to efficiently produce recombinant proteins in mammalian cell culture is critical for the production of both research agents and commercial products. Several approaches and host vector systems for the production of recombinant proteins have been reviewed (Kaufman, Genetic Engineering, Principles and Methods, vol. 9, J. K, Setlow, ed., Plenum Press, New, York, 1987; Warren et al., Recombinant DNA Technology and Applications, A. Prokop, R. Bajpai and C. Ho, eds., McGraw Hill, New York, 1990). These systems include use of high copy episomal vectors such as bovine papillomavirus (Howley et al., Methods in Enzymology, vol. 101, Academic Press, New York, 1983), amplifiable vectors such as those containing the dihydrofolate reductase gene (Kaufman,supra), the asparagine synthetase gene (Andrulis, Molecular Cell Genetics, vol. 17, 1985) or the ornithine decarboxylase gene (McConlogue, Gene Transfer Vectors for Mammalian Cells. 1987) or strong constitutive promoters such as the simian virus 40 promoter (Mulligan et al., Science. vol. 209, pp. 1422-1427, 1980) or the human cytomegalovirus major early promoter (Boshart et al., Cell, vol. 41, pp. 521-530, 1985). All of these systems rely upon the levels of endogenous transactivators in the particular cell type to stimulate transcription of the promoters used to construct the expression vectors.
An alternative approach to high level production would be to engineer cells with a specific transcriptional activator or transactivator. If the transactivator has a specific target promoter, then the target promoter can be linked to a gene of interest and inserted into the engineered cell. The amount of target protein produced from that cell would depend on several parameters. First, the inherent specific activity of the transactivator will be a factor in the amount of transcription from the target promoter. In addition, the amount of transactivator produced by the target cell will affect the amount of transactivation. For instance, in Chinese hamster ovary cells (CHO) there is a low level of endogenous glucocorticoid receptor/transactivator present. Transfection of a plasmid that requires the glucocorticoid receptor/transactivator results in very little expression from that plasmid. However, if the cells are first engineered to express high levels of the glucocorticoid receptor/transactivator, then high level expression from the same plasmid is obtained (Israel et al., Nuc. Acids Res., vol. 17, pp. 4589-4606, 1989). Therefore, the amount of transactivation depends on the amount of transactivator in the cell. The amount of transactivator will depend on the promoter used to drive expression of the transactivator and the site of the integration of the cassette in the host cell. Thirdly, the amount of target vector in a particular cell will influence how many copies will be transactivated. The site of integration of the target promoter may also play a role in the expression of the activated promoter.
Another important concern is the specificity of the transactivator. If the transactivator interacts with several endogenous cellular promoters, then it would be expected that those promoters will also be transactivated in the engineered host cell. This may or may not be a desired situation depending on what those genes are. A possible effect of using a promiscuous transactivator is that its binding to the endogenous promoters effectively lowers its free concentration in the cell, perhaps titrating out the amount available for transactivation of the targeted promoter.
Just because a promoter shows high level expression under transient conditions does not indicate that it will be useful for production of proteins under stable conditions. An example of which is the human cytomegalovirus (HCMV) immediate early (IE) promoter which is one of the strongest promoters used in the field for transient expression (Foecking et al., Gene, 45; pp. 101-145, 1986; Hippenmeyer et al., Poultry Science 70, pp. 982-992, 1991). In addition some viral genes do not have the expected regulation once integrated into the cell genome such as the thymidine kinase gene of herpes simplex virus (HSV) (Silver et al., Molecular and Cellular Biology 5; pp. 518-528, 1985).
Therefore, although E.P. application 8802149.5 shows that a cell line in which VP16 (also known as Vmw65, VF65 or alpha-TIF) is produced leads to transactivation of the target promoter under transient conditions and although Post et al, (Cell 24, pp. 555-565, 1981) showed that the immediate early (IE)175 promoter (also known as ICP4), when resident in the genome of a cell, can be transactivated by virus infection, neither show that when both VP16 and the target promoter operably linked to a gene of interest are integrated in the same cell that high level transactivation occurs and high level production of protein results.
There is a need in the art for cell lines and systems which can be used with a variety of genes to achieve stable, high level, recombinant protein production.
The invention contains a method of producing cell lines for high level expression of a gene product. The method comprises the steps of: cotransfecting a cell with a first construct which causes the cell to express herpes simplex virus transactivating protein VP16 and a second construct comprising a selectable resistance gene to a first selectable agent; selecting cells which are resistant to the first selectable agent; screening the cells which are resistant to the first selectable agent for cells which express VP16; cotransfecting the cell which expresses herpes simplex virus transactivating protein VP16 with third and fourth constructs, the third construct comprising herpes simplex virus gene promoter operably linked to a gene of interest, the fourth construct comprising a selectable resistance gene to a second selectable agent; selecting cells which are resistant to the second selectable agent; and screening cells which are resistant to the second selectable agent for expression of the gene product of said gene of interest.
In another embodiment of the invention are cell lines which express high levels of a gene product. The cell lines are made by the process of: cotransfecting a cell with a first construct which causes the cell to express herpes simplex virus transactivating protein VP16 and a second construct comprising a selectable resistance gene to a first antibiotic; selecting cells which are resistant to the first antibiotic, screening the cells which are resistant to the first antibiotic for cells which express VP16; cotransfecting the cell which expresses herpes simplex virus transactivating protein VP16 with third and fourth constructs, the third construct comprising a herpes simplex virus IE gene promoter operably linked to a gene of interest, the fourth construct comprising a selectable resistance gene to a second antibiotic; selecting cells which are resistant to the second antibiotic; and screening cells which are resistant to the second antibiotic for high expression of the gene product of said gene of interest.
Another embodiment of the invention in which a cell line expresses a high level of a gene product is provided. The cell lines comprise: a first construct which causes the cell to express herpes simplex virus transactivator protein VP16; a second construct comprising herpes simplex virus IE gene promoter operable linked to a gene of interest; a third and fourth construct each comprising a selectable resistance gene to an antibiotic.
In the above embodiments all types of herpes simplex virus IE promoters are intended, preferably from herpes simplex virus-1, preferably IE175 (also known as ICP4) or IE110 (also known as ICP0). The cell lines are preferably derived from baby hamster kidney (BHK-21) or Chinese Hamster Ovary (CHO-DUKX-B11 or DG44).
All types of VP16 are intended to be included in the invention but preferably VP16 from herpes simplex virus 1. The VP16 gene and protein from HSV-2 are anticipated to work as well as the VP16 gene and protein from HSV-1 due to the strong similarity in amino acid sequence between the two (Greaves et al Journal of Virology 65, pp. 6705-6713 1991; Cress et al Gene, 103, pp. 235-238, 1991).
The use of the cell lines in discovering compounds that interfere with the transactivation of the IE promoters of HSV by the VP16 molecule, is also intended. It is envisioned that a cell line that stably secretes a recombinant protein due to transactivation of an IE promoter by VP16 can be incubated with chemicals or natural products or unspecificied mixtures of chemicals or natural products. If the incubation of the cell with these compounds leads to a decrease in the amount of recombinant protein produced by the cell, then the compound is potentially acting at the VP16-mediated transactivation step of the recombinant protein expression. These compounds would then be tested to see if they interfere with the expression of a recombinant protein from a cell line in which expression was not under control of VP16. Those compounds that interfere with expression of the VP16-mediated transactivation and not expression in other-systems are potential anti-HSV agents.
It is anticipated that cell lines engineered to express VP16 will be useful for the passage and maintainence of mutants of HSV that lack a functional VP16 (Werstuck et al., Journal of Virology. vol. 64, pp. 984-991). The DNA of HSV which lacks a functional VP16 gene or protein can be transfected into a cell line that expresses VP16 from the integrated DNA constructs. The VP16 protein will transactivate the IE promoters of the HSV DNA leading to production of virus particles. Mutants of HSV that lack functional VP16 may be promising candidates for a vaccine strain.
The invention is intended to cover cell lines whether the cell lines secrete or do not secrete the gene product but preferably the cell lines secrete the gene product.
It is an object of the invention to provide a method of producing a cell line for high level expression of a gene product.
It is another object of the invention to provide cell lines which express a high level of a gene product.
It is still another object of the invention to provide the art with cell lines which can be used for the large scale production of any gene product of interest.
It is still another object of the invention to provide the art with means to propagate VP16-minus herpesvirus mutants.
It is still another object of the present invention to provide an assay for discovering anti-viral compounds.
It is an advantage of the invention to provide the art with stable cell lines that which can express a high level of a gene product for a period exceeding 5 months.
It is still another advantage of this invention in that a method is provided which allows isolating cell lines that produce higher expression levels of a gene product per cell per day faster than the art and the cell line is stable for a period exceeding 5 months. Many other objects and purposes of the invention will be clear from the following descriptions of the invention.